Our long-term goal is to understand the regulation of cell growth by cAMP-dependent protein kinase (PKA) by an in-depth investigation of the structure, function and expression of the regulatory (R) and catalytic (C) subunits of PKA. PKA is a key receptor protein for cAMP signals, transducing the signals inside mammalian cells. Importantly, there are two isoforms, type I (PKA-I) and Type II (PKA-II), and expression of these PKA isoforms can change dramatically during cell development, differentiation and transformation. PKA-I and PKA-II contain different R subunits, RI and RII, respectively, but share a common C subunit. We have investigated the following: (1) The functional distinction between PKA-I and PKA-II by structure-function analysis using site-directed mutagenesis and overexpression/suppression of the R and C subunit genes; (2) The cross-talk of PKA with other signaling pathway components in accordance with alteration of PKA expression through overexpression/suppression of the R and C subunit genes; and (3) The cooperative/antagonistic function of PKA with other cellular regulatory proteins and signal receptors by biochemical and immunocytochemical analysis. For functional distinction analysis of PKA isozymes, we targeted the RIa subunit with antisense. LS-174T colon carcinoma and LNCap prostate carcinoma cells mainly express RIa, RIIa and Ca subunits of PKA and the RIIb subunit at undetectable level. The loss of RIa by the antisense resulted in rapid increase in RIIb and PKA-IIb holoenzyme. Pulse-Chase experiments demonstrated that RIIb protein increased its half-life 5.5 fold in antisense treated cells (control, RIIb t <sub>1/2</sub>, 2h; antisense treated, RIIb t <sub>1/2</sub>, 11h). Thus, RIIb in the holoenzyme complex is stabilized, exhibiting an increased half-life. Through this biochemical adaptation, in the antisense-treated cancer cells, the ratio of PKA-I to PKA-II changes to become similar to that of normal cells. These studies rigorously demonstrated that RIa/PKA-I expression promotes cell proliferation, and that the compensatory stabilization of RIIb protein represents an important biochemical mechanism of RIa antisense that ensures depletion of PKA-I leading to sustained inhibition of tumor cell growth. In summary, our results indicate that RIa/PKA-I is a positive regulator of cell growth while RIIb/PKA-IIb (RIIb- containing PKA) is a growth inhibitory protein, supporting our hypothesis (Cho-Chung, Cancer Res. 50:7093-7100, 1990). Our results also imply that other signaling pathways that regulate cell growth most likely involve PKA chaperone. - Cancer, Differentiation, cAMP-dependent protein kinase, growth arrest, cyclic AMP analog, antisense, Apoptosis, EGF receptor,